Summary There is a critical gap in our knowledge regarding the molecular mechanisms that control signaling events during intestinal homeostasis. This gap represents a barrier to scientific progress because, until it is addressed, an explanation for diseases resulting from developmental disorders in the gut will continue to be beyond our understanding. Furthermore, this gap in the knowledge hinders progress in the development of therapies to promote recovery of the intestine following injury or damage. Our long-term goal is to identify molecular mechanisms involved in epithelial homeostasis. The objective of this proposal is to identify roles for physiological ROS generation from gut- specific NADPH oxidases (Nox enzymes) in normal gut development. Based on our preliminary data, our central hypothesis is that ROS generated by Nox1 in the intestinal epithelia functions to stimulate host gene regulatory events within the intestinal stem cell (ISC) microenvironment. In addition, we have discovered that colonization of the metazoan gut with specific strains of symbiotic bacteria induces the generation of ROS within enterocytes. Thus, we also hypothesize that contact of specific members of the microbiota (and candidate probiotic agents) with intestinal cells induces NADPH oxidases to generate ROS which then act as transducers of bacterial signals into host gene regulatory events that influence homeostasis in the metazoan gut. The rationale for this hypothesis is based on established reports that ROS, especially H2O2 function as signaling molecules to modulate protein activity through the oxidation of sensor cysteine residues within regulatory proteins. In our preliminary data, we show that both intestinal-specific Nox1- null mice, and Drosophila with diminished Nox1 levels have altered intestinal physiology. Importantly, we also show that lactobacilli, which are commonly employed as candidate probiotic agents, are potent inducers of Nox1 cellular ROS generation in intestinal epithelial cells, and are potent inducers of cell proliferation by a Nox1-dependent mechanism. Based on these compelling preliminary data generated by our research group, the central hypothesis will be tested in three specific aims: 1) Identify the function of NADPH oxidases in intestinal epithelium development and homeostasis, 2) Identify the function of NADPH oxidases in intestinal epithelium regeneration following injury, and 3) Identify the influence of bacterial-induced and NADPH oxidase- dependent ROS generation on intestinal healing following injury. Our approach will employ an intestinal epithelial cell-specific deficient nox1 (B6.Nox1IEC) mouse, and a highly innovative genetically tractable Drosophila model whose biology can be manipulated to a far greater extent than mammalian models. Also, there is striking conservation in the molecular mechanisms of intestinal development between Drosophila and mammals. The outcomes of these investigations will have a positive impact on public health because of direct implications to idiopathic intestinal and systemic immune and developmental disorders and provides a springboard to the development of preventative interventions for these conditions.